This invention relates to a biocompatible, implantable material which is absorbed naturally in vivo with minimal immunological reaction, and to the methods for its production. More particularly, this invention relates to a novel collagenous bone matrix useful as an allogenic or xenogenic implant for use as an osteogenic device, as a bone particle coating for implantable prostheses, as a delivery vehicle for the in vivo sustained release of protein, and as a substratum for growth of anchorage-dependent cells.
A biocompatible, implantable material that can be resorbed in vivo could be used to promote conductive bone growth, induce osteogenesis when combined with an osteoinductive protein, provide a substratum for in vivo or in vitro growth of anchorage-dependent cells, or serve as a carrier for the sustained release of, for example, a therapeutic drug or antibiotic. Such a material must be biocompatible, that is, must not induce an immunogenic or continued inflammatory response in vivo. Its physical structure must allow cell infiltration, and it must have an in vivo resorption time appropriate for its function.
The potential utility of an osteogenic device capable of inducing endochondral bone formation in vivo has been recognized widely. It is contemplated that the availability of such devices would revolutionize orthopedic medicine, certain types of Plastic surgery, and various periodontal and craniofacial reconstructive procedures.
The developmental cascade of bone differentiation induced by the implantation of demineralized/bone matrix is well documented in the art (Reddi, 1981, Collagen Rel. Res. 1:209-226). Although the precise mechanisms underlying the phenotypic transformations are unclear, it has been shown that the natural endochondral bone differentiation activity of bone matrix can be dissociated into two principle components: a soluble proteinaceous component responsible for osteogenic activity, and an insoluble collagenous matrix (residue serves as a carrier for bone induction). The soluble osteoinductive protein components are then reconstituted with inactive residual collagenous matrix to restore full bone inducing activity (Sampath et al., Proc. Natl. Acad. Sci. USA 7599-7603 (1981). Recently, the protein factors hereafter referred to as osteogenic protein (OP) responsible for inducing osteogenesis have been purified, expressed in recombinant host cells, and shown to be truly osteoinductive when appropriately sorbed onto allogenic demineralized bone powder. (U.S. Pat. application Ser. No. 179,406 now U.S. Pat. No. 4,968,590.
Studies have shown that while osteoinductive proteins are useful cross species, the collagenous bone matrix generally used for inducing endochondral bone formation is species specific (Sampath and Reddi (1983) PNAS 80:6591-6594). Implants of demineralized, delipidated, extracted xenogenic bone matrix as a carrier in an in vivo bone induction system invariably has failed to induce osteogenesis, presumably due to inhibitory or immunogenic components in the bone matrix. However, even the use of allogenic bone matrix in osteogenic devices may not be sufficient for osteoinductive bone formation in many species. For example, allogenic, subcutaneous implants of demineralized, delipidated monkey bone matrix is reported not to induce bone formation in the monkey. (Asperberg et al., J. Bone Joint Suro. (Br) 70-B:625-627 (1988)).
U.S. Pat. No. 4,563,350, published Jan. 7, 1986, discloses the use of trypsinized bovine bone matrix as a xenogenic matrix to effect osteogenic activity when implanted with extracted partially purified bone inducing protein preparations. Bone formation is said to require the presence of at least 5%, and preferably at least 10%, non-fibrillar collagen in the disclosed matrix. The authors claim that removal of telopeptides which are responsible in part for the immunogenicity of collagen preparations is more suitable for xenogenic implants.
EPO 309,241 (published 3/29/89, filed 9/22/88, priority 9/25/87) discloses a device for inducing endochondral bone formation comprising an osteogenic protein preparation, and a matrix carrier comprising 60-98% of either mineral component or bone collagen powder and 2-40% atelopeptide hypoimmunogenic collagen.
Deatherage et al., (1987) Collagen Rel. Res. 7:2225-2231, purport to disclose an apparently xenogenic implantable device comprising a bovine bone matrix extract that has been minimally purified by a one-step ion exchange column and reconstituted highly purified human Type-I placental collagen.
In order to repair bone defects in orthopedic reconstructive surgery, biomaterials based on collagens, minerals/ceramics, polymers and metal implants are being used as implants. These biomaterials are known to support healing by conduction but do not induce new bone. The current state of the art of materials used in surgical procedures requiring conductive bone repair, such as the recontouring or filling in of osseous defects, is disclosed by Deatherage (1988) J. Oral Maxillofac. Surg. 17:395-359. All of the known implant materials described (hydroxylapatite, freeze-dried bone, or autogenous bone grafts) have little or no osteoinductive properties. The ability to induce osteogenesis is preferred over bone conduction for most procedures.
U.S. Pat. No. 4,795,467 discloses a bone repair composition comprising calcium phosphate minerals (preferable particle size of 100-2,000.mu.) and atelopeptide, reconstituted, crosslinked, fibrillar collagen. It purports to be a non-antigenic, biocompatible, composition capable of filling bony defects and promoting bone growth xenogenically.
U.S. Pat. No. 4,789,663 discloses using xenogenic collagen from bone and/or skin to effect conductive bone repair by exposing the defect to fresh bone, wherein the collagen is enzymatically treated to remove telopeptides, and is artificially crosslinked.
In order to enhance bone ingrowth in fixation of orthopedic prostheses, porous coatings on metallic implants are being employed. Although the surface chemistry of porous coatings plays a role in bone conduction, it appears bone has higher tensile and shear strength and higher stiffness at the porous coating - bone interface. The need to provide a "biological anchor" for implanted prostheses, particularly metallic implants, is well documented in the art. The state of the art of prosthetic implants, disclosed by Specter (1987) J. Arthroplasty 2:163-177, generally utilizes porous coated devices, as these coats have been shown to promote cellular ingrowth significantly.
Recently the art also has sought to increase cellular ingrowth of implanted prostheses by coating their surfaces with collagen preparations. For example, EPO 169,001 (published 1/22/86) claims a collagen-coated prosthesis wherein the coat comprises a purified, sterile, non-immunogenic xenogenic collagen preparation from bone or skin. The collagen is preferably atelopeptide collagen. The coating is formed by dipping the prosthesis into a suspension of collagen, or forming a collagen sheet that is wrapped about the prosthesis.
U.S. Pat. No. 4,812,120 discloses a prosthetic device comprising a metal core over which are applied successive polymer layers. The outer layer comprises a biopolymer having protruding collagen fibrils. The protruding fibrils are subject to damage upon implantation of the device. Increased surface area and pore size in a matrix has been shown to enhance cell attachment and growth of anchorage-dependent cells in vitro.
Efficient in vitro growth of mammalian cells is often limited by the materials used as the substratum or "scaffold" for anchorage-dependent cells. An optimal matrix for this purpose must be physiologically acceptable to the anchorage dependent cells, and it must also provide a large available surface area to which the cells can attach. GB U.S. Pat. No. 2,178,447, published Feb. 11, 1987, discloses a fibrous or porous foam matrix comprising open or closed form fibers, with a pore size on the order of 10-100.mu.m (matrix height is 50-500.mu.). The fiber network is generated as a sheet which must then be modified if different scaffold shapes are desired. Strand et al. (Biotechnology and Bioengineering, V. 26, 503, 1984) disclose microcarrier beads for use as a matrix for anchorage dependent cells in a matrix perfusion cell culture. Bead materials tested were DEAE or polyacrylamide. Surface area available was 250-300 cm.sup.2/ g and required a cell innoculation of 10.sup.6 cells/ml. U.S Pat. No. 4,725,671 claims collagen fiber membranes suitable for cell culture, comprising soluble atelopeptide collagen fibers that are dried and preferably cross-linked.
The art has sought sustained release vehicles with known, reliable "release" rates. Effective carriers must be biocompatible, water-insoluble, capable of trapping or otherwise holding the therapeutic agent of interest for a required time, and must have a resorption time in vivo that mimics the desired release rate of the agent. Collagens are attractive carriers for clinical use, primarily because of their biocompatible and biodegradable properties. The carriers are generally formulated into "sponge-like" structures by solubilizing or dispersing collagen and then solidifying the solution so that monofilaments are captured in a generally, random, open-structured array. The solvent is then removed, and the molecules chemically crosslinked to maintain the open-structure and render the carrier water insoluble. The therapeutic compound is preferably mixed with the collagen in solution prior to solidification.
The structures can be made in a variety of shapes. For example, EPO 230,647, published Aug. 5, 1987, discloses structures formed as micropellets. The structures can also be made in sheet form (U.S. Pat. No. 4,703,108, published,, Oct. 27, 1987), rods or tubes (see, for example, EPO 069,260, published Jan. 12, 1983, EPO 170,979, published Feb. 12, 1986, and U.S 4,657,548, published Apr. 14, 1987), or beads. U.S. Pat. No. 4,837,285, published Jun. 6, 1989, discloses a composition for wound dressings or drug delivery systems made of porous beads formed by freeze-drying microdroplets containing the agent of interest and solubilized or dispersed Type I or Type III collagen. The microdroplets are then slowly lyophilized or air-dried to form beads which are then crosslinked.
Unfortunately, the high "fiber-forming" property of collagen can interfere with the formation of uniform and homogeneous solutions, making efficient synthesis of appropriate carrier matrices difficult. In addition, the use of crosslinking agents (e.g., glutaraldehyde) may have adverse biological effects, such as cell cytotoxicity (Cooke, et al. British J. Exo. Path. 64. 172, 1983).
It is an object of this invention to provide a biocompatible, in vivo biodegradable bone matrix, implantable in a mammalian host with little or no significant inhibitory or immunogenic response. Another object is to provide a biocompatible, in vivo biodegradable matrix capable in combination with an osteoinductive protein of producing endochondral bone formation in mammals, including humans. Still other objects are to provide a superior material for coating implantable prosthetic devices, to increase the cellular ingrowth into such devices, to provide a biocompatible, in vivo biodegradable matrix for use as a carrier of sustained-release pharmaceutical compositions, wherein the resorption rate of the matrix can be adjusted to match that of the pharmaceutical agent, and to provide a biocompatible, in vivo biodegradable matrix capable of acting as a scaffold or substratum for anchorage-dependent cells, wherein the surface area available for cell attachment can be adjusted. Yet another object of the invention is to provide a method for the production of such matrix material.
These and other objects and features of the invention will be apparent from the description, drawings, and claims that follow. As used herein, "bone collagen matrix" or "bone matrix" is intended to mean stripped, cleaned and demarrowed, pulverized, delipidated bone that has been demineralized and protein extracted with guanidine hydrochloride or an equivalent extractant. "Implantable", as used herein, includes surgical introduction as well as topical application, and introduction by injection.